The present invention relates the formation of both linear and non-linear liquid gradients from liquids having very small density differences, and to the formation of gradients having different reagents in different levels of the gradient. The invention further relates to the formation of sets of gradients in parallel. These are particularly useful for fractionation of cellular or subcellular particles from biological samples.
Liquids having gradients of temperature, concentration, density and color have been previously prepared. Liquid density gradients have been used for many years, for a variety of purposes, in a number of different industries. The inventor has numerous publications and patents regarding certain aspects of gradient formation and use including: Anderson, N. G. Mechanical device for producing density gradients in liquids. Rev. Sci. Instr. 26: 891-892, 1955; Anderson, N. G., Bond, H. E., and Canning, R. E. Analytical techniques for cell fractions. I. Simplified gradient elution programming. Analyt. Biochem. 3: 472-478, 1962; Anderson, N. G., and Rutenberg, E. Analytical techniques for cell fractions. A simple gradient-forming apparatus. Anal. Biochem. 21: 259-265, 1967; Candler, E. L., Nunley, C. E., and Anderson, N.G. Analytical techniques for cell fractions. VI. Multiple gradient-distributing rotor (B-XXI). Anal. Biochem. 21: 253-258, 1967.
A variety of other methods for making density gradients have been developed, and Bock, R. M. and Ling, N. S., Analyt. Chem 26, 1543 (1954), and Morris, C. J. O. R, and Morris, P., Separation Methods in Biochemistry, Pitman Publishing, 2nd ed., 1976, have reviewed many of these. Only one of these methods allows gradients to be made from multiple solutions, each having a different combination of reagents (Anderson, et al., xe2x80x9cAnalytical Techniques for Cell Fractions. I. Simplified Gradient Elution Programmingxe2x80x9d, Analytical Biochemistry 3: 472-478 (1962).) Innovations that are more recent include the use of pumps and pistons, which are differentially controlled by microprocessors, e.g., the Angelique gradient maker (Large Scale Proteomics Corp. Rockville, Md.). Gradients may also be generated during centrifugation by sedimenting a gradient solute such as cesium chloride or an iodinated x-ray contrast medium such as Iodixanol(copyright). Additional references discussing such methods are mentioned in the References section below. The large number indicates the importance of the problem of gradient making.
Density gradients are used to make two basic types of separations. The first separates particles based on sedimentation rate (rate-zonal centrifugation), in which particles are separated based on the size and density and to a lesser extent their shape. The particles will sediment farther if centrifuged for a longer period of time. The second method separates particles based on isopycnic banding density, in which particles reach their equilibrium density level, and do not sediment further with continued centrifugation.
One object of the present invention is to produce segments of a liquid density step gradient which differ predictably in their properties, and in the identity and concentration of reagents present in individual segments.
It is a further object to make all aspects of sample and gradient production and centrifugation identical so that the gradients are reproducible.
It is another object of the present invention to prepare a liquid having plural gradients of different types.
It is yet another object of the present invention to mix gradient components to prepare intermediate gradient components for accurately generating fine differences in the gradient.
It is yet a further object of the present invention to use liquids with small density differences to lessen the irregularities in forming a gradient.
Another object of the present invention is to make the sedimentation through a gradient an analytical process for analytical particle recovery from the liquid gradient and analytical measurement of particles in the gradient.
A further object of the present invention is a means for producing individual segments of the gradient.
It is still another object of the present invention to use a microprocessor to control and schedule an automatic pipetting system to produce multiple gradient segments in which the difference between each gradient segment is small and suitable for producing narrow zones which may be evened out by timed diffusion.
It is another object of the present invention to control the temperature of all steps in the gradient production and gradient recovery process and to avoid temperature induced mixing.
It is a further object of the present invention to provide means for making linear and complex non-linear gradients.
It is another object of the present invention to provide means for subfractionating biological particles by sedimenting them through zones of reagents with which the particles interact.
It is a further object of the present invention to construct complex gradients in which gradient stability is conferred by an inert component while superimposed gradient reagents extract or separate certain particles or constituents from the particles sedimenting through the gradient, leaving the extracted constituents at their extraction levels.
The present invention achieves these objectives by using a large number of gradient components where fine differences are achieved by mixing one gradient component with another to prepare an intermediate gradient component. Additional intermediate gradient components may also be similarly made. Even if the exact concentration is uncertain, the range must be correct. This technique assures that inversions and other irregularities do not occur. This technique is also readily automatable and can prepare multiple gradients in the same solution. Of particular benefit is the inclusion of a reagent with specialized properties within a particular region of the gradient to enhance separation and recovery of the sample particles.
Preferred uses are for separation and quantification of biological particles in a reproducible manner.